Autophagy-dependent cell cycle arrest in esophageal cancer cells exposed to dihydroartemisinin

Abstract

Background: Dihydroartemisinin (DHA), a derivate of artemisinin, is an effective antimalarial agent. DHA has been shown to exert anticancer activities to numerous cancer cells in the past few years, while the exact molecular mechanisms remain to be elucidated, especially in esophageal cancer. Methods: Crystal violet assay was conducted to determine the cell viability of human esophageal cancer cell line Eca109 treated with DHA. Tumor-bearing nude mice were employed to evaluate the anticancer effect of DHA in vivo. Soft agar and crystal violet assays were used to measure the tumorigenicity of Eca109 cells. Flow cytometry was performed to evaluate ROS or cell cycle distribution. GFP-LC3 plasmids were delivered into Eca109 cells to visualize autophagy induced by DHA under a fluorescence microscope. The mRNA and protein levels of each gene were tested by qRT-PCR and western blot, respectively. Results: Our results proved that DHA significantly reduced the viability of Eca109 cells in a dose-and time-dependent manner. Further investigation showed that DHA evidently induced cell cycle arrest at the G2/M phase in Eca109 cells. Mechanistically, DHA induced intracellular ROS generation and autophagy in Eca109 cells, while blocking ROS by an antioxidant NAC obviously inhibited autophagy. Furthermore, we found that telomere shelterin component TRF2 was down-regulated in Eca109 cells exposed to DHA through autophagy-dependent degradation, which could be rescued after autophagy was blocked by ROS inhibition. Moreover, the DNA damage response (DDR) was induced obviously in DHA treated cells. To further explore whether ROS or autophagy played a vital role in DHA induced cell cycle arrest, the cell cycle distribution of Eca109 cells was evaluated after ROS or autophagy blocking, and the results showed that autophagy, but not ROS, was essential for cell cycle arrest in DHA treated cells. Conclusion: Taken together, DHA showed anticancer effect on esophageal cancer cells through autophagy-dependent cell cycle arrest at the G2/M phase, which unveiled a novel mechanism of DHA as a chemotherapeutic agent, and the degradation of TRF2 followed by DDR might be responsible for this cell phenotype.

Knock-Down of Mucolipin 1 Channel Promotes Tumor Progression and Invasion in Human Glioblastoma Cell Lines

“…Autophagy can block apoptosis (59) but can be also permissive to senescence entry in cells resistant to apoptosis (55,59). Autophagic-dependent G2/M cell cycle phase arrest upon intracellular free radical generation was reported in esophageal cancer cells (41). Similarly, in siTRPML1 U251 cells, we demonstrated that the autophagy induction and the accumulation of cells in G2/M phase promoted cellular senescence as evidenced by increased SA-β-Gal + cells, compared to siGLO cells.…”

“…Autophagy-dependent G2/M cell cycle arrest was reported by Ma et al on intracellular free radical generation in esophageal cancer cells (41). Thus, cell cycle was evaluated at 24, 48, and 72-h post-transfection in siTRPML1 T98 and U251 cells compared to siGLO control cells (Figure 3A).…”

Progress in Redirecting Antiparasitic Drugs for Cancer Treatment

“…The mammalian target of the rapamycin (mTOR) pathway plays an inhibitory role in autophagy, and targeting the mTOR pathway to induce autophagy has become a cancer treatment therapy. 49 Wang et al reported that DHA functions as an mTOR inhibitor in Hela cells, thus inducing autophagy. 32 Interestingly, a novel ART derivative, SM1044, also shows the autophagy-inducing property in a different manner.…”

Artemisinins as Anticancer Drugs: Novel Therapeutic Approaches, Molecular Mechanisms, and Clinical Trials

“…Some evidence showed that autophagy induced by ARS and DHA was to protect cancer cells (Jia et al, 2014;Jiang et al, 2018), whereas autophagy induced by DHA was to kill cancer cells (Qu et al, 2017). Other evidence of autophagydepended ferroptosis, cell cycle arrest and cell apoptosis induced by ARTs and ARTs-induced autophagy sensitized chemotherapy drugs to enhance the cell death demonstrated the killing effect (Feng et al, 2014;Zhang Z. S. et al, 2015;Cheng et al, 2018;Du et al, 2019;Ma et al, 2020), whereas autophagy inhibitor enhanced the anticancer property of ARTs, indicating the protecting effect of autophagy (Chen S. S. et al, 2015). The molecular mechanisms of ART-induced autophagy involved accumulation of ROS, which activated JNK pathway in pancreatic cancer cells (Jia et al, 2014), or stimulating de novo synthesis of ceramide and CaMKK2-AMPK-ULK1 axis, which in turn cause the occurrence of autophagy in diffuse large B-cell lymphomas (Cheng et al, 2018), or increasing the expression of death-associated protein kinase 1 (DAPK1), reducing the interaction of beclin1 with bcl-2 and promoting the interaction of beclin1 with PI3KC3 in cholangiocarcinoma (Thongchot et al, 2018).…”

“…Some evidence showed that autophagy induced by ARS and DHA was to protect cancer cells ( Jia et al, 2014 ; Jiang et al, 2018 ), whereas autophagy induced by DHA was to kill cancer cells ( Qu et al, 2017 ). Other evidence of autophagy-depended ferroptosis, cell cycle arrest and cell apoptosis induced by ARTs and ARTs-induced autophagy sensitized chemotherapy drugs to enhance the cell death demonstrated the killing effect ( Feng et al, 2014 ; Zhang Z. S. et al, 2015 ; Cheng et al, 2018 ; Du et al, 2019 ; Ma et al, 2020 ), whereas autophagy inhibitor enhanced the anticancer property of ARTs, indicating the protecting effect of autophagy ( Chen S. S. et al, 2015 ).…”